Materials Synthetic Biology

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Synthetic biology, the science of engineering complex biological systems with novel functions, is increasingly fascinating researchers across disciplines who gather to design functional biological assemblies in a rational and systematic manner. Although initial success stories were based on reprogramming prokaryotic and lower eukaryotic cells, the design of synthetic mammalian gene circuits is becoming increasingly popular because it promises to foster novel therapeutic opportunities in the not-so-distant future. Here, we discuss the latest generation of mammalian synthetic biology devices assembled to form complex synthetic gene networks, such as regulatory cascades, logic evaluators, hysteretic circuits, epigenetic toggle switches, time-keeping components, drug discovery tools, and "cell phone" units. We further highlight how such circuits could be interconnected to achieve higher-order control networks such as synthetic hormone-like communication systems in animals or synthetic ecosystems with dynamic interspecies crosstalk. © 2009 Elsevier Ltd. All rights reserved.

The emergence of synthetic biology is holding great hopes for providing solutions to the unmet needs of humankind. This review article describes how synthetic biology can deliver on this promise in the field of drug discovery by providing novel opportunities throughout the entire drug discovery process. Synthetic biology tools enable disease mechanisms and target identification to be elucidated and also provide avenues to discover small chemotherapeutic molecules or design novel biopharmaceuticals. Furthermore, synthetic biologists can design cost-effective microbial production processes for complex natural products, which could help overcome global drug shortages. These impressive advances have been achieved in only a few years, and are an indicator for the potential of synthetic biology. © 2009 Elsevier Ltd. All rights reserved.

Adjustable and reversible transgene expression systems enabling precise control of metabolic pathways and tunable production of specific target proteins have been essential for conditional reprogramming of mammalian cells to achieve progress in basic and applied bioengineering disciplines. Most of the currently available transgene control modalities have been designed to be responsive to clinically licensed pharmacologically active drugs which were expected to prevail in future clinical trials yet raised concerns about side effects when administered long term at subclinical doses. We have chosen vitamin H, also known as biotin, to control target gene transcription in mammalian cells in a potentially side effect-free manner. BirA, the Escherichia coli repressor of the biotin biosynthesis operon, was fused to the Herpes simplex transactivation domain to generate a biotin-dependent transactivator (BIT), which, in the presence of biotin, binds and activates chimeric target promoters (PBIT) harboring BirA-specific operator sites 5′ of a minimal promoter. Biotin-inducible transgene expression was functional in a variety of rodent, monkey and human cell lines, showed excellent adjustability and reversibility in transgenic Chinese hamster ovary cell lines, provided precise product gene control in standard bioreactor cultures and enabled dose-dependent vitamin H control of a human glycoprotein in mice. The combination of a side effect-free inducer, precise and reversible transcription tunability and broad functionality in different cell types as well as in entire animals represents a unique asset for the use of biotin-inducible transgene control in future gene therapy, tissue engineering and biopharmaceutical manufacturing scenarios. © 2009 Elsevier Inc. All rights reserved.

Targeted delivery of therapeutic transgenes into specific cells remains a highly relevant challenge for tissue engineering and future gene-based therapies. We have designed streptavidin-pseudotyped lentiviral particles which upon coupling with biotinylated magnetic carbon-coated cobalt nanoparticles could be guided by magnetic fields to site-specifically transduce desired target cells in culture as well as in mice. Magnetic patterns projected onto monolayer cultures were replicated by fluorescent cells following targeted transduction by magnetic lentiviral particles engineered for constitutive expression of the green fluorescent protein (GFP). Even after intravenous injection into mice magnetic GFP-transgenic lentiviral particles could be guided to a preferred transduction site in the animal using a magnetic field. Magnet-guided transgene delivery producing desired patterns of transduced cell populations may enable the design of defined tissue topologies and provide site-specific transduction of therapeutic transgenes for cell-specific interventions in future gene and cancer therapies. © 2009 Elsevier B.V. All rights reserved.

The release of volatile ethylene and acetaldehyde characterizes the metabolic state and quality of fruit. We have designed and implemented a hybrid dual-channel catalytic-biological sensor system, which is able to quantify both volatiles in situ. This sensor system consists of a mammalian cell line engineered for constitutive expression of an Aspergillus nidulans-derived biosensor which triggers quantitative reporter gene expression in the presence of volatile acetaldehyde. Ethylene, oxidized to acetaldehyde using a Wacker-based process, can be quantified by the same transgenic sensor cell line. Differential profiling of reporter gene transcription by the sensor system revealed the relative concentrations of both volatile metabolites and enabled correct assessment of fruit quality as shown for fresh, old and rotten apples. Functional combination of catalytic processes with biosensor technology is able to precisely capture the metabolic state of food and may foster novel insight into biochemical food quality assessment as well as the design of synthetic control circuits detecting and preventing food spoilage. © 2009 Elsevier B.V. All rights reserved.

Electric signal processing has evolved to manage rapid information transfer in neuronal networks and muscular contraction in multicellular organisms and controls the most sophisticated man-built devices. Using a synthetic biology approach to assemble electronic parts with genetic control units engineered into mammalian cells, we designed an electric power-adjustable transcription control circuit able to integrate the intensity of a direct current over time, to translate the amplitude or frequency of an alternating current into an adjustable genetic readout or to modulate the beating frequency of primary heart cells. Successful miniaturization of the electro-genetic devices may pave the way for the design of novel hybrid electro-genetic implants assembled from electronic and genetic parts. © 2009 The Author(s).

Functionally well-characterized modular transcription units represent the genetic repertoire for the design of synthetic gene networks operating inside individual mammalian cells. Interconnection of specialized cells to multicellular assemblies that could execute complex computational functions requires synthetic signaling systems, which process and synchronize metabolic information between mammalian cells. In this study we have designed a metabolite-controlled inter-cellular signaling device consisting of a human sender cell line stably engineered for constitutive expression of the human liver-type arginase and a transgenic receiver cell line harboring a synthetic circuit, which produced a human glycoprotein in response to l-arginine levels in the culture medium. Quantitative characterization of the system components enabled precise prediction of l-arginine degradation and product gene expression kinetics and showed that two independent transgenic cell lines could functionally inter-operate to form a metabolite-controlled device which is able to precisely time desired target gene expression. Synthetic gene circuits modulating the transfer of metabolic information from a sender to a receiver cell line may enable the design of synthetic hormone systems supporting communication across multicellular assemblies. © The Royal Society of Chemistry 2009.

Drug-dependent dissociation or association of cellular receptors represents a potent pharmacologic mode of action for regulating cell fate and function. Transferring the knowledge of pharmacologically triggered protein-protein interactions to materials science will enable novel design concepts for stimuli-sensing smart hydrogels. Here, we show the design and validation of an antibiotic-sensing hydrogel for the trigger-inducible release of human vascular endothelial growth factor. Genetically engineered bacterial gyrase subunit B (GyrB) (ref. 4) coupled to polyacrylamide was dimerized by the addition of the aminocoumarin antibiotic coumermycin, resulting in hydrogel formation. Addition of increasing concentrations of clinically validated novobiocin (Albamycin) dissociated the GyrB subunits, thereby resulting in dissociation of the hydrogel and dose- and time-dependent liberation of the entrapped protein pharmaceutical VEGF 121 for triggering proliferation of human umbilical vein endothelial cells. Pharmacologically controlled hydrogels have the potential to fulfil the promises of stimuli-sensing materials as smart devices for spatiotemporally controlled delivery of drugs within the patient. © 2008 Macmillan Publishers Limited. All rights reserved.

Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In Mycobacterium tuberculosis EthR binds to a specific operator (OethR) thereby repressing ethA and preventing EthA-catalyzed conversion of the prodrug ethionamide, which increases the resistance of the pathogen to this last-line-of-defense treatment. We have designed a synthetic mammalian gene circuit that senses the EthR-O ethR interaction in human cells and produces a quantitative reporter gene expression readout. Challenging of the synthetic network with compounds of a rationally designed chemical library revealed 2-phenylethyl-butyrate as a nontoxic substance that abolished EthR's repressor function inside human cells, in mice, and within M. tuberculosis where it triggered derepression of ethA and increased the sensitivity of this pathogen to ethionamide. The discovery of antituberculosis compounds by using synthetic mammalian gene circuits may establish a new line of defense against multidrug-resistant M. tuberculosis. © 2008 by The National Academy of Sciences of the USA.